Project Summary/Abstract Critical issues emerging from current attempts at inducing transplantation tolerance in the clinic include the need for biomarkers that predict and diagnose tolerance as well as for the identification of barriers to tolerance induction and maintenance. Our preliminary observations have led us to the realization that the durability of the tolerant state is variable and can be unpredictably breached by Listeria infections. The overall goal of the proposed studies Is to determine a mechanistic basis for how Infections can either erode or transiently abrogate robust tolerance after It has been stably established. We propose 3 specific aims: Specific Aim 1: Our investigations to date have determined that the abrogation of established tolerance by Listeria infection is mechanistically different from the prevention of tolerance induction, and that the combination of IL-6 and IFNU is necessary and sufficient to abrogate established tolerance. We will conduct mechanistic studies to determine how the combination of IL-6 and IFNB abrogates established tolerance. Specific Aim 2: Approximately 25% of tolerant recipients infected with Listeria do not reject their grafts. However, this retained state of tolerance acquires susceptibility to reversal by PDL-1 blockade, whereas tolerance prior to Listeria infection does not. We will determine the mechanistic bases for these observations, and test whether other infections such as Staphylococcus aureus and influenza, that cannot precipitate rejection in recipients with established tolerance, can nonetheless erode the quality of tolerance and make it increasingly susceptible to reversal. Specific Aim 3: Our investigations have recently revealed an unexpected resilience of the state of transplantation tolerance. Infection with Listeria only transiently subsumes the tolerant state but tolerance spontaneously returns after the infection is cleared. We will define why and how tolerance is spontaneously restored when infection with Listeria is cleared. We anticipate that these studies will provide novel and clinically relevant insights into how tolerance to allografts is maintained, abrogated and potentially restorable. Our studies may also lead to the identification of biomarkers for the diagnosis and assessment of the quality of tolerance, and to the definition of therapies that can maintain and boost the quality of tolerance. Finally, we speculate that studies on how tolerance is transiently lost but spontaneously re-established might reveal fundamental principles of how T cell responses are regulated not only in transplantation tolerance but also in autoimmunity and in the recurrence of cancers.